The cross-linking reagent copper-o-phenanthroline complex (Cu(OP)2) specifically caused a decrease in the amount of the 30-kDa ADP/ATP carrier in bovine submitochondrial particles associated predominantly with formation of a 60-kDa protein consisting of a cross-linked dimer of the carrier. However, Cu(OP)2 had no effect on mitochondria. The transport of ADP via the carrier through submitochondrial particle membranes was found to be inhibited in parallel with the progress of intermolecular cross-linking. Analysis of the cross-linked site showed that a disulfide bridge was formed only between two Cys56 residues in a pair of the first loops facing the matrix space. The transport inhibitor bongkrekic acid, which locks the m-state conformation of the carrier, had no effect on disulfide bridge formation catalyzed by Cu(OP)2, but carboxyatractyloside, which locks the c-state conformation by acting from the cytosolic side, completely inhibited the cross-linking. These results show that the ADP/ATP carrier functions as a dimer form, and a pair of the first loops protrudes into the matrix space in the m-state, but possibly intrudes into the membrane in the c-state. Thus, it is suggested that a pair of the first loops acts as a gate and that its opening and closing are regulated by their translocation.

Purpose. The effect of AST-120, an oral adsorbent, on oxidative stress in the systemic circulation in chronic renal failure (CRF) was examined and the potential role of indoxyl sulfate (IS), an uremic toxin adsorbed by AST-120, in inducing the formation of reactive oxygen species (ROS) in the vascular system was studied, in vitro and in vivo. Materials and methods. The level of oxidized albumin, a marker for oxidative stress in the systemic circulation was determined by HPLC, as previously reported. The mRNA levels of TGF-b 1 and Oat1 were measured by quantitative RT-PCR. The IS induced ROS generation in cultured human umbilical vein endothelial cells (HUVECs) was estimated using a fluorescence microplate reader.Results. An increase in the ratio of oxidized to unoxidized albumin was determined using 5/6 nephrectomized rats (CRF rats) compared to a control group. The ratio was significantly reduced in the group that received AST-120 of 4 weeks, suggesting that AST-120 inhibits oxidative stress in CRF. An anti-oxidative effect of AST-120 was also observed in CRF rats with a similar renal function. The ratio of oxidized albumin was correlated with serum IS levels in vivo. The same relationship was also observed in CRF rats with the continued administration of IS. In addition, IS dramatically increased the generation of ROS in both a dose-and time-dependent manner in HUVEC, suggesting that accumulated IS may play an important role in enhancing intravascular oxidative stress. Conclusion. We propose that AST-120 reduces IS concentrations in the blood that induces ROS production in endothelial cells, thereby inhibiting the subsequent occurrence of oxidative stress in the systemic circulation in renal failure.

To characterize the transport mechanism mediated by the mammalian mitochondrial ADP/ATP carrier (AAC), we tried to express bovine heart mitochondrial AAC (bhAAC) in Saccharomyces cerevisiae. The open reading frame of the bhAAC was introduced into the haploid strain WB-12, in which intrinsic AAC genes were disrupted. Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane. For improvement of bhAAC expression in WB-12, we introduced DNA fragments encoding chimeric bhAACs, in which the N-terminal region of the bhAAC extending into the cytosol was replaced by the corresponding regions of the type 1 and type 2 yeast AAC isoforms (yAAC1 and yAAC2). These transformants grew well, and the amounts of the chimeric bhAACs in their mitochondria were as high as that of yAAC2. The carriers expressed showed essentially the same ADP transport activities as that of AAC in bovine heart mitochondria.

ABSTRACT:The absorption characteristics of temocapril were investigated using Caco-2 cells, and the esterases expressed in Caco-2 cells were identified. Temocapril was almost completely hydrolyzed to temocaprilat during transport across Caco-2 cells. Hydrolysis experiments of temocapril in Caco-2 cell 9000g supernatant (S9) and brush-border membrane vesicles showed that temocapril was mainly hydrolyzed within the cells after uptake, after which the temocaprilat formed was transported to both the apical and basolateral surfaces. In native polyacrylamide gel electrophoresis by detection of hydrolase activity for 1-naphthylbutyrate, Caco-2 cell S9 showed a band with high esterase activity and another band with extremely low activity. The proteins in the major and minor bands were identified as carboxylesterase-1 (hCE-1) and carboxylesterase-2 (hCE-2). The abundant expression of hCE-1 in Caco-2 cells was supported by reverse transcription-polymerase chain reaction. In the normal human small intestine, hCE-2 is abundantly present, although the human liver expresses much higher levels of hCE-1 and lower levels of hCE-2. The expression pattern of carboxylesterases in Caco-2 cells is completely different from that in human small intestine but very similar to that in human liver. Since the substrate specificity of hCE-1 differs from that of hCE-2, it is suggested that the prediction of human intestinal absorption using Caco-2 cell monolayers should be performed carefully in the case of ester-and amide-containing drugs such as prodrugs.

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